Human Pathology (2005) 36, 724 – 731
www.elsevier.com/locate/humpath
Poly(adenosine diphosphate-ribose) polymerase 1
expression in malignant melanomas from photoexposed
areas of the head and neck regionB
Stefania Staibano MD, PhDa,f,*, Stefano Pepe MDb,f,
Lorenzo Lo Muzio MD, PhDd, Pasquale Somma MDa, Massimo Mascolo MDa,
Giuseppe Argenziano MDe, Massimiliano Scalvenzi MDc,f, Gaetano Salvatore VDh,
Gabriella Fabbrocini MDc,f, Guido Molea MDf,g, Angelo Raffaele Bianco MDb,f,
Chiara Carlomagno MDb, Gaetano De Rosa MDa,f
a
Pathology Section, Department of Biomorphological and Functional Sciences, University Federico II of Naples,
80127 Naples, Italy
b
Department of Molecular and Clinical Endocrinology and Oncology, University Federico II of Naples, 80127 Naples, Italy
c
Department of Dermatology, University Federico II of Naples, 80127 Naples, Italy
d
Department of Surgical Sciences, University of Foggia, Italy
e
Department of Dermatology, S.U.N. University, 80127 Naples, Italy
f
Cooperative Melanoma Group, Federico II University, 80127 Naples, Italy
g
Department of Plastic Surgery, University Federico II of Naples, 80127 Naples, Italy
h
Department of Medicine, University Federico II of Naples, 80127 Naples, Italy
Keywords:
Cutaneous melanoma;
PARP-1;
Prognosis
Summary The family of the poly(adenosine diphosphate-ribose) polymerase (PARP) proteins is directly
involved in genomic stability, DNA repair, and apoptosis by DNA damage. In this study, we evaluated
the role of PARP-1 in melanoma and its prognostic importance. We studied by immunohistochemistry
and Western blot analysis PARP-1 expression in a selected series of 80 primary melanoma of the head
and neck region. The results were correlated with tumor thickness and patient’s outcome. A follow-up
of at least 3 years was available. Fifteen cases of benign melanocytic nevi were used as controls.
Normal melanocytes showed only scattered, focal nuclear positivity and were considered as negative
for PARP-1 expression by immunohistochemistry (score, 0). Thirty cases of melanoma (37.5%)
showed nuclear expression of PARP-1 in both radial and vertical growth phases. Western blot analysis
showed the presence of a high signal for full-length PARP-1 only in the cases with high
immunohistochemical (nuclear) expression of protein (score, ++/+++) in both radial and vertical
growth phase. A significant correlation was present between PARP-1 expression in vertical growth
phase and the thickness of tumor lesion ( P = .014); all but one tumor measuring less than 0.75 mm
B
This study was supported by grants from the Italian Ministry of University, Scientific and Technologic Research (Rome, Italy) and Associazione Italia
Ricerca sul Cancro (Milan, Italy).
T Corresponding author.
E-mail address: [email protected] (S. Staibano).
0046-8177/$ – see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.humpath.2005.04.017
PARP-1 and malignant melanomas
725
showed no or low PARP-1 expression. No correlation was found between PARP-1 expression in radial
growth phase and tumor thickness ( P = .38, data not shown). These data suggest that PARP-1
overexpression is a potential novel molecular marker of aggressive cutaneous malignant melanoma and
a direct correlation between PARP-1–mediated inhibition of the apoptosis and biologic behavior of
cutaneous malignant melanoma.
D 2005 Elsevier Inc. All rights reserved.
1. Introduction
In the last 2 decades, the incidence of all skin cancers is
increasing in most Western countries and in Australia.
Cutaneous malignant melanoma (CMM) death incidence is
increasing faster than most of other skin malignancies at
present, with an incidence that has tripled during the last 40
years and continues to grow [1- 4].
In the United States, 32 868 white subjects have been
diagnosed with melanoma during 1975 through 1990,
covering approximately 10% of the American population
[5]. In Italy, the incidence of CMM in the hospital referral
population is steadily rising, and at present, this tumor
represents one of the few causes of premature death with
unfavorable trends [1,6,7].
The most significant prognostic factor (at the time of
the diagnosis) is the extent of tumor invasion expressed by
thickness rather than anatomic structure [2]. Patients with
melanoma measuring 1.00 mm or less have a 5-year
survival of 96%; thicker tumors are associated with a poor
prognosis. In the last decade, there has been a positive
trend of thin lesions at diagnosis [8]; however, some of
these cases, often cured by the excisional biopsy with
conservative surgical margins, show an unfavorable outcome that is unpredictable based on the classic clinicopathologic parameters [9].
Individual risk factors play an important part in the
development of malignant melanoma [10]. The most
important phenotypic risk factors are the number of total
body acquired melanocytic nevi and the occurrence of
previous epithelial skin cancer [11-15].
Solar damage is the major environmental causal factor in
all skin cancers, and intermittent intense exposures to
sunlight and/or severe sunburn are also the most important
environmental risk factors for CMM [9].
A history of sunburn up to 12 years is one of the primary
sun-related factors associated with an increased risk for
CMM [16].
Even if the carcinogenic effect of UV rays has been
attributed predominantly to short-wavelength (290 -320 nm)
UV radiation (UV-B), a carcinogenic role has been reported
also for the long-wavelength (320 - 400 nm) UV light
(UV-A) [17,18].
UV-A radiation exerts penetrating effects on the skin,
causing DNA damage and increasing the risk for cancer.
Recently, basic research on UV radiation showed that DNA
repair via different pathways causes a cascade of cellular
phenomena ranging from induction of pigmentation to
immunomodulation, acid radical defense, apoptosis, and
oncogene expression [19].
The members of the poly(adenosine diphosphate-ribose)
polymerase (PARP) family proteins are directly involved in
genomic stability, DNA repair, and cell death triggered by
DNA damage. However, their potential role in carcinogenesis has not been well evaluated [20].
We evaluated the immunohistochemical expression of
PARP-1 in a series of CMM from photoexposed areas, and
the correlation between PARP-1 expression and tumor
thickness and the patient’s outcome. Western blot (WB)
analysis was also performed on a limited number of cases to
confirm the immunohistochemical data.
The aim of the study was to evaluate the role of PARP-1
in tumor progression and its prognostic importance in
cutaneous melanomas.
2. Materials and methods
2.1. Selection of cases
All the cases of primary cutaneous melanomas diagnosed between January 1985 and December 1998 at the
Precancerous Unit, Department of Dermatology, University
Federico II of Naples, Italy, were reviewed. All the cases
of CMM that have arisen in photoexposed areas of the
head and neck region without a hereditary history of skin
cancer and/or prior physical or chemical predisposing
environmental factors and with a follow-up of at least
3 years were considered suitable for the present analysis.
The paraffin-embedded blocks of these selected patients,
stored in the archive of the Pathology Section, Department
of Biomorphological and Functional Sciences, University
Federico II of Naples, Italy, were used for the immunohistochemical determination of PARP-1 expression. Moreover, 5 specimens of human normal skin were obtained
from patients who had undergone surgical procedures for
reconstructive surgery (with the informed consent of the
donors) and 15 cases of benign melanocytic nevi
(5 junctional, 5 compound, and 5 intradermal nevi) were
used as controls.
2.1.1. Immunohistochemistry
Four-micrometer serial sections from routinely formalin-fixed, paraffin-embedded blocks were cut for each
case of cutaneous melanomas, melanocytic nevi, and
726
S. Staibano et al.
Table 1
Expression of PARP in CMM and follow-up
Case Age Sex
(y)
New staging PARPr PARPv Follow-up
system,
(y)
AJCC
1
2
3
4
5
6
7
8
9
10a
11a
12a
13a
14a
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40a
41a
42a
43a
44a
45a
46a
47a
48a
49a
50a
51a
52a
53
54
55
V1.00
V1.00
V1.00
V1.00
V1.00
V1.00
V1.00
V1.00
V1.00
V1.00
V1.00
V1.00
V1.00
V1.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
1.01-2.00
2.01-4.00
2.01-4.00
2.01-4.00
58
36
67
45
50
49
42
43
43
49
40
41
39
37
66
65
56
49
48
39
47
51
53
56
51
45
53
67
54
34
44
43
71
34
43
51
50
39
29
42
37
65
43
44
18
22
24
32
30
38
37
40
40
36
39
Female
Male
Male
Female
Female
Female
Male
Female
Male
Male
Male
Male
Male
Female
Female
Female
Male
Male
Male
Female
Female
Male
Male
Male
Female
Female
Male
Male
Male
Male
Female
Female
Female
Male
Female
Female
Male
Female
Female
Male
Female
Male
Male
Female
Female
Male
Male
Male
Female
Female
Male
Male
Male
Male
Male
0
0
0
0
+
0
0
+
0
0
+
+
++
0
++
0
0
0
0
0
+++
0
+
0
0
+
0
0
0
+
0
0
0
0
0
0
0
+++
0
0
+++
0
+++
+
+
0
0
0
0
+
0
0
0
0
0
+
+
+
+
+
0
+
+
0
0
+
+
+++
+
+++
0
0
0
+
0
+++
+
++
++
+
++
++
+
0
++
++
0
++
0
0
++
0
+++
+
++
+++
+
+++
+
++
0
+
+
0
+
+
0
+
0
0
12
12
12
12
11
11
10
9
9
7
7
7
6R
6
12 N
12
12
12
12
12
12 N, M, D
11
11
11
10
10
10
10
10
9
9
9
9
9
9
9
9
9N
9
7
7 R, N
7
7N
6
4
4
4
4
4
4
3
3
12
12
11
Table 1
56
57
58
59
60
61
62
63
64
65
66
67a
68a
69a
70a
71a
72a
73a
74a
75a
76a
77a
78a
79a
80a
50
48
45
43
53
47
54
50
69
54
53
42
37
45
49
32
38
38
32
40
46
50
24
30
32
continued
Female
Female
Male
Female
Female
Male
Male
Male
Female
Male
Male
Female
Female
Female
Female
Male
Female
Male
Male
Male
Female
Male
Female
Male
Female
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
2.01-4.00
+
0
+++
0
+
++
+
+
0
0
0
+++
+++
0
+++
+++
++
0
++
0
+
0
+
0
0
++
++
+++
+
++
+++
++
++
0
+
+
+++
+++
+
+++
+++
+++
+
+++
+
+++
0
++
+
+
11
11
11 N,
10
10
10 N
10
10
10
10
9
9 N,
9 N,
9
9 N,
7N
6N
3
3 N,
3
3N
2
2
2
2
M, D
M, D
M
M, D
M
Abbreviations. PARPr, radial growth; PARPv, vertical growth;
R, recidivation; N, lymph node metastasis; M, metastasis; D, death
for tumor.
a
WB.
normal skin, and were mounted on poly-l-lysine–coated
glass slides.
Deparaffinized sections were boiled 3 times for 3 minutes
in a 10 3 mol/L sodium citrate buffer (pH 6.0) as an antigen
retrieval method. To prevent the nonspecific binding of the
antibody, sections were preincubated with nonimmune
mouse serum (1:20; Dakopatts, Hamburg, Germany) diluted
in phosphate-buffered saline–bovine serum albumin (1%) for
25 minutes at room temperature. After quenching of
endogenous peroxidases with 0.3% hydrogen peroxide in
methanol, followed by 2 rinses with Tris-HCl buffer, the
sections were incubated with the anti–PARP-1 primary
antibody (PARP F2, mouse monoclonal IgG2a, raised against
a recombinant protein corresponding to amino acids
764-1014 mapping at the carboxyl terminus of PARP of
human origin; Santa Cruz Biotechnology, Inc, Santa Cruz,
Calif) diluted 1:50 overnight at 48C. The standard streptavidin-biotin-peroxidase complex technique, using sequential
20-minute incubation with biotinylated linking antibody and
peroxidase-labeled streptavidin (DAKO labeled streptavidinbiotin-complex kit horse radish peroxidase; Carpinteria,
Calif), was performed. 3,3V-Diaminobenzidine (3-3V diaminobenzidine tetrachloride; Vector Laboratories, Burlingame,
Calif) was used as a substrate chromogen solution for the
development of the peroxidase activity. Hematoxylin was
used for nuclear counterstaining; then, the sections were
PARP-1 and malignant melanomas
727
The samples were subjected to sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (14% polyacrylamide)
under reducing conditions. After electrophoresis, proteins
were transferred to nitrocellulose membrane (Immobilon;
Millipore, Bedford, Mass); complete transfer was assessed
using prestained protein standards (Bio-Rad). The membranes
were treated for 2 hours with blocking solution (5% nonfat
powdered milk in 25 mmol/LTris, pH 7.4; 200 mmol/L NaCl;
0.5% Triton X-100; Tris-buffered saline [TBS]–Tween 20),
and then, the membranes were incubated for 12 hours at 48C
with the primary anti-PARP antibody. After washing with
TBS–Tween 20 and TBS, membranes were incubated with
the horseradish peroxidase–conjugated secondary antibody
(1:5000) for 1 hour (at room temperature), and the reaction
was detected with enhanced chemiluminescence system
(Amersham Life Science, Piscataway, NJ).
2.3. Statistical analysis
Fig. 1 A, Strongly immunoreactive for PARP-1 (cutaneous superficial spreading malignant melanoma, worse prognosis, vertical
growth phase) (avidin-biotin complex technique, original magnification 150). B, Absent immunostaining for PARP-1 in a case of
cutaneous superficial spreading malignant melanoma (vertical
growth phase, node negative, disease-free 10 years after surgery)
(avidin-biotin complex technique, original magnification 250).
mounted and coverslipped with a synthetic mounting
medium (Entellan; Merck, Darmstadt, Germany).
As positive controls, the immunoreactivity of normal
squamous epithelium next to the tumor was evaluated.
Negative controls were performed using an antibody with
irrelevant specificity but with the same isotype as the
primary antibody and were included in each staining run.
The nuclear expression of PARP-1 was evaluated semiquantitatively according to an arbitrary scale as follows:
0 (b5% of positive cells), + (V25%), ++ (26%-50%), and
+++ (N50% of positive cells).
PARP-1 expression, scored in 4 classes (negative, 0; low,
+; moderate, ++; strong, +++), was grouped in 2 categories
(negative/low, 0/+, and moderate/strong, ++/+++). The
thickness of the tumor lesions was coded according to
new staging system by the American Joint Committee on
Cancer (AJCC, 2001; T1, V1.00 mm; T2, 1.01-2.00 mm;
T3, 2.01-4.00 mm; T4, N4.00 mm) [21,22].
The correlation between PARP-1 expression and the
thickness of the tumor lesion was evaluated by the v 2 test.
Disease-free survival (DFS) was calculated from the date
of surgery to the date of the first locoregional recurrence
or distant metastases. DFS curves were drawn by the KaplanMeier method, and the statistical significance of the differences was calculated by log-rank test at univariate analysis.
All analyses have been performed using the S-Plus 2000
software (MathSoft Inc, Cambridge, UK).
3. Results
3.1. Patients
2.2. Western blot analysis
Following the aforementioned selection criteria, 80 cases
of CMM were considered suitable for the analysis. The
Frozen tissues from 10 compounds and intradermal
melanocytic nevi and from 30 cases of CMM were
homogenized directly into lysis buffer containing 50 mmol/L
4 -(2-hydroxyethyl)-1-piperazineethanesulfonic acid,
150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L ethyleneglycotetraacetic acid, 10% glycerol, 1% Triton X-100
(weight/volume, 1:2), 1 mmol/L phenylmethylsulfonyl
fluoride, 1 lg aprotinin, 0.5 mmol/L sodium orthovanadate,
and 20 mmol/L sodium pyrophosphate (Sigma, St Louis,
Mo) and were clarified by centrifugation at 14 000g for
10 minutes. Protein concentrations were estimated using a
modified Bradford assay (Bio-Rad, Melville, NY).
Fifty micrograms of total protein extracts were boiled in
Laemmli buffer for 5 minutes before electrophoresis.
Fig. 2 Western blot showing full-length line positive in A and B
(A, node positive, brain metastasis at 3 years after surgery; B, death
for disease 5 years after surgery) and line positive in C and D, with
a light presence of cleaved form (good prognosis at 8 and 12 years
after surgery) (normalized for b-tubulin).
728
S. Staibano et al.
Table 2 PARP expression in radial and vertical growth
phase of CMM with poor prognosis
New staging
system,
AJCC
Cases Cases
with poor
prognosis
PARPr/PARPv Follow-up
V1.00
1.01-2.00
14
38
2.01-4.00
28
++/+++
++/+++
+++/+++
+++/+++
+++/+++
+++/+++
++/+++
+++/+++
+++/+++
++/+++
+/+++
–
N4.00
0
1
1
1
1
2
3
2
1
1
1
1
0
R
N
N, M, D
R, N
N
N, M, D
N
N, M
N
N, M
N
0
Abbreviations. PARPr, radial growth; PARPv, vertical growth; R,
recidivation; N, lymph node metastasis; M, metastasis; D, death
for tumor.
study population consisted of 43 men and 37 women, with a
mean age of 44.5 years (range, 18-71 years). All the patients
had undergone surgical treatment with curative intention at
the Plastic Surgery Department of the University Federico II
of Naples, Italy. The extent of invasion was assessed on
hematoxylin-eosin–stained sections from formalin-fixed,
paraffin-embedded tissue according to new staging system
by the AJCC (2001) [21,22]: 14 (17.5%) patients had less
than 1.00 mm; 38 (47.5%), between 1.01 and 2.00 mm; and
28 (35%), between 2.01 and 4.00 mm. No patients had
CMM of greater than 4.00 mm.
(37.5%) showed nuclear expression (between + and +++) of
PARP-1 in both radial and vertical growth phase (Fig. 1A).
Several cases showed absent immunostaining for PARP-1
(Fig. 1B); cases were characterized by node negative and
good behavior. Among the 30 cases of CMM examined by
WB analysis (Fig. 2), the presence of high signal for fulllength PARP-1 was found only in the cases with high
immunohistochemical (nuclear) expression of the protein
(score, ++/+++) in both radial and vertical growth phase.
Overexpression of full-length PARP-1 and low levels of
cleaved PARP-1 were found in CMM with high immunohistochemical expression of PARP-1 restricted to the cells
of the vertical phase of growth; the cases of CMM with
low/absent expression of PARP-1 in the vertical growth
phase and absent PARP-1 expression in the radial growth
phase showed a low/moderate amount of both cleaved
and full-length PARP-1.
A significant correlation was present between PARP-1
expression in vertical growth phase and the thickness of the
tumor lesion ( P = .014); all but one tumor measuring less
than 1.00 mm showed no or low PARP-1 expression. No
correlation was found between PARP-1 expression in radial
growth phase and tumor thickness ( P = .38, data not shown).
3.2. PARP-1 expression in normal melanocytes
Normal skin specimens showed positivity for PARP-1
only in epithelial cells of basal and, less frequently,
parabasal layers; normal melanocytes showed only scattered, low nuclear immunostaining, with a ratio of about
1:5 melanocytes, and this value was defined as bnormal
PARP-1 expressionQ (score, 0).
3.3. PARP-1 expression in melanocytic nevi
By immunohistochemistry, positive expression of
PARP-1 was observed in all the 15 cases of melanocytic
nevi as strong nuclear staining in up to 30% of melanocytes
(mean positivity, 20% of melanocytes), and this was defined
as blow intensityQ (score, +).
The WB analysis showed the presence of both full-length
and cleaved PARP-1 expression in all the 10 cases examined
(5 compound and 5 intradermal nevi).
3.4. PARP expression in malignant melanomas
The expression of PARP-1 in the vertical (invasive)
and in the radial growth phase of CMM correlated with
follow-up data is reported in Table 1. Thirty cases of CMM
Fig. 3 DFS according to Breslow level (A) and PARP
expression (B).
PARP-1 and malignant melanomas
3.5. Patient’s outcome
Overall, 15 patients relapsed: 7 patients at locoregional
lymph nodes, 1 at skin, 1 at locoregional lymph nodes and
skin, and 6 patients presented concomitant locoregional
lymph nodes and distant metastases (Table 2).
As expected, DFS was affected by tumor’s thickness;
among the 15 relapsed patients, 9 were between 2.01 and
4.00 mm, 5 were between 1.01 and 2.00 mm, and only 1 had
1.00 mm or less. Thus, the actual proportion of relapse was
32.1% (9/28 patients) for T3 cases, 13.1% (5/38 patients) for
T2, and only 7% (1/14 patients) for T1 patients.
Interestingly, PARP-1 overexpression demonstrated to be
a strong predictor of relapse; all the 15 relapsed patients had
tumors with ++/+++ PARP-1 expression in vertical growth
phase, and 9 of them also had ++/+++ PARP-1 expression in
radial growth phase.
Patients with ++/+++ PARP overexpression (either in
vertical or in radial growth phase) showed a significantly
short DFS than patients with low or negative PARP-1
overexpression (Fig. 3A and B).
4. Discussion
Long-term exposure to sunlight causes photoaging,
immunosuppression, and skin cancer. Failure of the DNA
repair processes constitutes one of the major molecular
events underlying UV-related skin carcinogenesis. The UV
radiation normally induces pyrimidine dimer formation,
which leads either to DNA repair or apoptosis. However,
UV radiation may determine multiple mutations in genes
regulating apoptosis and DNA repair, leading to the
uncontrolled cellular proliferation.
In the present study, we found a significant correlation
between the nuclear overexpression of PARP-1 and the
vertical (invasive) growth phase of melanomas, whereas the
neoplastic melanocytes of the radial phase were characterized by a slight nuclear expression of the PARP-1.
As it is well-known, activation of PARP-1 is an
immediate cellular reaction to DNA strand breakage
induced by alkylating agents, ionizing radiation, or
oxidants. The resulting formation of protein-coupled poly
(adenosine diphosphate-ribose) facilitates survival of proliferating cells under conditions of DNA damage, probably
via its contribution to DNA base-excision repair [23].
Furthermore, recent data indicate that PARP-1 acts as a
negative regulator of genomic instability in cells under
genotoxic stress [24]. The p53 tumor suppressor protein is
activated by a variety of cellular insults, including UV
radiation, to become a transcription factor for downstream
markers such as the cyclin kinase inhibitor p21CIP1/WAF1
or caspase transactivation, which cleaves PARP-1 as an
early step in apoptosis [25,26]. In fact, during apoptosis,
CPP32 (caspase 3) cleaves the 116-kd death substrate
PARP into a stable 85-kd fragment containing the carboxyl
729
terminal and a 25-kd fragment [27,28]. The degradation of
nuclear PARP-1 then is suggestive of caspase-mediated
early apoptotic events.
Although the involvement of the PARP family proteins
in the control of genomic stability, in DNA repair, and in the
regulation of apoptosis program has been outlined before,
their potential role in carcinogenesis has not been well
evaluated [29].
In bronchogenic tumorigenesis, for example, cigarette
smoke augments asbestos-induced bronchogenic carcinoma
in a synergistic manner by mechanisms that are not
established. One important mechanism may involve alveolar
epithelial cell injury resulting from oxidant-induced DNA
damage that subsequently activates PARP-1 [30].
The findings of the present study suggest that the
neoplastic progression toward the invasive (vertical) growth
phase of melanocytes in CMM is characterized by the loss
of cleavage of PARP-1, probably signaling an imbalance of
the apoptotic process in these cells and therefore predisposing them to acquire alteration(s) of other gatekeeping genes,
leading to further gain to aggressivity.
More interestingly, in our series of cases, the presence of
overexpression of full-length PARP-1 in both (radial and
vertical) growth phases was correlated with recurrence and/
or progression of the disease.
The overexpression of full-length PARP-1 in both radial
and vertical growth phase appears then as a promising new
marker of worse prognosis for CMM of photoexposed areas.
Previous studies showed a deregulation of the apoptotic
process in malignant melanomas. In particular, BCL-2
protein expression has been reported in most CMM [31].
As it is well-known, BCL-2 is the principal member of a
family of proteins with either positive or negative activity on
the apoptotic process [32,33].
It constitutes the prototype of the antiapoptotic proteins and has been found overexpressed in most human
malignancies [34].
To the best of our knowledge, our study represents the
first evidence of a direct correlation between the inhibition
of the apoptotic process (PARP-1–mediated) and the
biologic behavior of CMM. A strong reactivity with
BCL-2 antibody is observed in melanocytes of normal skin.
In nevocellular nevi, immunoreactivity gradually decreases
or even disappears toward the deeper dermal component. In
malignant melanomas of all stages and histological subtypes, the neoplastic cells express BCL-2 oncoprotein, the
most intense positivity being restricted to cells in the radial
growth phase. Expression of the protein in the great majority
of malignant melanomas seems to exclude its prognostic
significance in these tumors, even if cutaneous and lymph
node metastases of malignant melanomas have been found
often negative or only weakly and focally reactive for
BCL-2 [35,36]. The expression of BCL-2 oncoprotein by
malignant melanomas adds these neoplasms to a growing
list of tumors expressing this oncoprotein. BCL-2 in
malignant melanoma may play a role in tumor development
730
by sparing the cells from apoptotic death (and thereby
exposing those to secondary events) or through cooperation
with other oncogenes [37]. The lack of reactivity in
metastatic melanoma suggests that mechanisms other than
BCL-2 are involved in the survival and growth of metastatic
melanoma cells [38].
Moreover, the analysis of PARP-1 expression may offer
useful information concerning the pharmacological treatment of CMM.
Conventional anticancer drugs, in fact, kill susceptible
cells through induction of apoptosis. Alteration of the
pathways leading to apoptosis deficiency might represent
a potent mechanism conferring drug resistance. Recent
studies demonstrated that PARP-1 cleavage is strongly
reduced in highly cisplatin-resistant melanoma cells sublines [39]. In addition, metastatic malignant melanoma is
notoriously resistant to chemotherapeutic agents, but the
exact mechanisms involved in this drug resistance are still
unknown [40]. The imbalance of the apoptotic process
provides a broad cytoprotective mechanism to cancerous
cells, counteracting apoptosis induced by various chemotherapeutic drugs [37]. The survival advantage due to the
full-length PARP-1 hyperaccumulation in melanoma cells,
related to a loss of susceptibility to apoptosis and to defects
in checkpoint pathways, may be responsible for the
chemoresistance of this tumor.
Recently, it has been reported that the inhibition of
PARP-1 activity is a promising strategy to improve
the outcome of cytotoxic therapies in different tumor
models [41,42].
In conclusion, the findings of the present study indicate
that the analysis of PARP-1 expression in CMM may be
potentially relevant for implementation of closer follow-up
protocols and/or alternative therapeutic regimens, reiterating
the importance of deregulation of apoptosis as a critical
pathogenetic component of tumor progression, and identify
PARP-1 overexpression as a potential novel molecular
marker of aggressive neoplasia.
Acknowledgments
The authors thank Mr Armando Coppola, Mr Pasquale
Signoriello, and Ms Viviana Strazzullo for their precious
technical assistance and Mrs Patricia Reynolds for the
English editing of the manuscript.
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